WO2014013338A2 - Composés pour le traitement de la maladie d'alzheimer - Google Patents

Composés pour le traitement de la maladie d'alzheimer Download PDF

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WO2014013338A2
WO2014013338A2 PCT/IB2013/001994 IB2013001994W WO2014013338A2 WO 2014013338 A2 WO2014013338 A2 WO 2014013338A2 IB 2013001994 W IB2013001994 W IB 2013001994W WO 2014013338 A2 WO2014013338 A2 WO 2014013338A2
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subject
administering
pharmaceutically acceptable
compounds
neuroprotective
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PCT/IB2013/001994
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WO2014013338A3 (fr
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Jia Luo
Gregory R.J. Thatcher
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Sgc Pharma, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • AD Alzheimer's disease
  • a neurological disorder characterized by a progressive decline in cognitive function, synaptic dysfunction, and diffuse neuronal loss coupled with classic histopathological changes.
  • Global estimates put the prevalence at 26.6 million people in 2006, with an expected rise to 1 in 85 people by 2050 [Brookmeyer, R., et al. Alzheimers Dement, 2007, 3(3): 186-91].
  • 5.4 million people suffer from AD with an annual cost approaching $184 billion [Association, A. Alzheimer's and Dementia, 2011, 7: 208-244].
  • AD Alzheimer Dis Assoc Disord, 2009, 23(2): 165-70].
  • finding a treatment option that mitigates the clinical progression of the disease is one of the most important research goals in the modern era.
  • the invention provides methods of using compounds in the treatment of neurodegenerative diseases and disorders.
  • a method of providing neuroprotection via a GABA A -dependent or GABA A -independent pathway comprises administering a compound of Formula I to a subject in need thereof.
  • this method comprises administering thecompound GN-28 or GN-38 to a subject in need thereof.
  • neuroprotection proceeds via a GABAA-dependent pathway. In another embodiment, neuroprotection proceeds via a GABA A - independent pathway. Another embodiment comprises administering a compound of Formula I to the subject, wherein a compound of Formula I acts with reduced sedative effects.
  • Yet another embodiment comprises administering a compound of Formula I to the subject, wherein a compound of Formula I acts in a neuroprotective and/or neurorestorative manner with reduced sedative effects.
  • a method for treating a neurodegenerative disease comprising administering a compound of Formula I to a subject in need thereof, wherein the compound of Formula I is associated with reduced sedative effects in the subject.
  • One embodiment of this method comprises administering a compound of Formula GN-28 and/or GN-38.
  • a method for treating AD or an AD related disorder comprising administering a compound of Formula I to a subject in need thereof, wherein the compound of Formula I is associated with reduced sedative effects in the subject.
  • One embodiment of this method comprises administering a compound of Formula GN-28 and/or GN-38
  • Another aspect provided herein is a method of upregulating or activating NO/cGMP/CREB pathway, wherein the method comprises administering 4-methyl- 5-(2-nitroxyethyl)thiazole (GT1061), or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
  • GT1061 4-methyl- 5-(2-nitroxyethyl)thiazole
  • NO/cGMP/CREB pathway provides neuroprotective and neurorestorative effects.
  • this method of administering GT1061, or a pharmaceutically acceptable salt thereof the upregulation or activation of the NO/cGMP/CREB pathway provides anti- inflammatory effects.
  • GT1061 is in the hydrochloride (HC1) salt form. In another embodiment, GT1061 is in the maleate salt form.
  • the upregulation or activation of the NO/cGMP/CREB pathway lowers the levels of AD ⁇ protein in the brain of a subject.
  • the upregulation or activation of the NO/cGMP/CREB pathway lowers the levels of tau in the brain of a subject.
  • Another embodiment provided herein is a method of administering GT1061, or a pharmaceutically acceptable salt thereof, to a subject, wherein GT1061 elicits neuroprotective and/or neurorestorative effects without causing sedation.
  • a method for lowering the level of tau in the brain of a subject in need thereof comprising administering to the subject a therapeutically effective amount of GT1061 , or a pharmaceutically acceptable salt thereof.
  • a method for treating a neurodegenerative disease comprising administering GT1061 , or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein GT1061 does not cause sedative effects in the subject.
  • a method for treating AD or an AD related disorder comprising administering GT1061, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein GT1061 does not cause sedative effects in the subject.
  • FIG 1 shows preliminary experiments from structure activity relationship (SAR) analysis.
  • Figure 2 shows MZ derivatives protect neurons against OGD with varied dependence on GABA A .
  • Figure 3 shows MZ dervicatives decrease glutamate release with varied dependence of GABA A .
  • Figure 4 shows MZ derivatives protect neurons against ⁇ oligomers with dependence on GABA A .
  • Figure 5 shows MZ derivatives protect against OGD at pharmacologically relevant concentrations and when added hours after OGD.
  • Figure 6 shows MZ derivatives protect against NMDA with varied dependence on GABA A
  • Figure 7 shows MZ derivatives protect against extracellular glutamate with varied dependence on GABA A .
  • Figure 8 shows protection against high dose oligomeric ⁇ by CMZ and GN- 28 not dependent on GABA A receptor activity.
  • Figure 9 shows sedative action of CMZ observed in the accerated rotorod task is attenuated in MZ derivatives.
  • Figure 10 shows GT1061 retains neuroprotective activity of CMZ against OGD and AB oligomers.
  • Figure 11 shows GT1061 attenuates sedation compared to CMZ in RR
  • Figure 12 shows GT1061 reverses amnestic deficits in STPA induced by diverse agents.
  • Figure 13 shows GT1061 reverses scopolamine-induced deficit from 10 m prior to and 90 m after training.
  • Figure 14 shows GT1061 acutely reverses synapticdeficits in APP/PSl mouse model.
  • Figure 15 shows ten-week administration of GT1061 produces a trend towards increased performance in the RAWM.
  • Figure 16 shows ten-week administration of GT1061 reduces inflammation in APP/PSl mice.
  • Figure 17 shows ten-week administration of GT1061 decreases soluble ⁇ in APP/PSlmice.
  • Figure 18 shows GT1061 acutely reverses synaptic deficits in 3XTG mouse model.
  • Figure 19 shows ten-week administration of GT1061 reverses behavioral deficits in STPA in 3XTG mice.
  • Figure 20 shows ten-week administration of GT1061 increases PCRED in 3XTG mice.
  • Figure 21 shows ten-week administration of GT1061 decreases tau and AP in 3XTg mice.
  • This invention is directed toward compounds, as well as pharmaceutical compositions containing the compounds, for use in the treatment of
  • the invention pro ides compounds of the Formula I:
  • R 1 is H, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a substituted or unsubstituted Ci-24 aliphatic group, -(CH 2 )i_ 3 -aryl, or - (CH 2 )i_3-heteroaryl;
  • R 2 is selected from the group consisting of H, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Ci_24 aliphatic group, - OR a , halo, N(R a )(R b ), N 3 , -(CH 2 )i- 3 N 3 , -(CH 2 )i- 3 R a and -(CH 2 )i- 3 halo, wherein R a and R b , independently, are selected from the group consisting of H, Ci_ 3 alkyl, aryl, or heteroaryl, or R a and R b are taken together with the atom to which they are attached to from a 3 to 6 membered ring; and
  • R 1 and R 2 are not H.
  • R 1 is H, OH, -(CH 2 )i- 3 -OH, unsubstituted aryl, or -(CH 2 )i_ 3 -aryl;
  • R 2 is H, aryl, heteroaryl, -OR a , N(R a )(R b ), or -(CH 2 )i_ 3 R a , wherein aryl or heteroaryl can be optionally substituted with halo, OH, Ci_6 primary alcohol, Ci_6 alkyl, aryl, or heteroaryl, wherein R a and R b , independently, are selected from the group consisting of H, Ci_ 3 alkyl, aryl, or heteroaryl, or R a and R b are taken together with the atom to which they are attached to from a 3 to 6 membered ring; and
  • R 1 and R 2 are not H.
  • R 1 is H, OH, -(CH 2 )i 3-OH, or phenyl
  • R 2 is H, phenyl, heteroaryl, -OR a , N(R a )(R b ), or -(CH 2 )i_ 3 R a , wherein aryl or heteroaryl can be optionally substituted with halo, aryl, or heteroaryl, wherein R a and R b , independently, are selected from the group consisting of H, Ci_ 3 alkyl, aryl, or heteroaryl, or R a and R b are taken together with the atom to which they are attached to from a 3 to 6 membered ring; and
  • AD related disorder includes, but is not limited to, frontal lobe dementia, Pick's disease, Parkinson disease with dementia, corticobasal degeneration, argyrophilic grains disease, progressive supranuclear palsy, subacute sclerosing panencephalitis, multiple system tauopathy dementia, and familial Gerstmann-Straussler-Scheinker Disease.
  • neurodegenerative disorder includes, but is not limited to, mild cognitive impairment (MCI), Down syndrome, Lewy body dementia, vascular dementia, and any AD related disorder, as listed above.
  • MCI mild cognitive impairment
  • Down syndrome Lewy body dementia
  • vascular dementia vascular dementia
  • AD related disorder as listed above.
  • neuronalestorative effects is defined as the maintenance of neuronal cells that would otherwise be destined to die as a result of a prior neuronal insult or neurodegenerative disease.
  • neurodegenerative disease As used herein, the term “neurorestorative effects” is defined as the maintenance of neuronal cells that would otherwise be destined to die as a result of a prior neuronal insult or neurodegenerative disease.
  • neuroprotective effects is defined as the restoration of cells that are not dead but that exist in a seriously dysfunctional state. For example, atrophied cholinergic and cortical neurons in the early stages of Alzheimer's disease are not dead yet but are seriously dysfunctional.
  • compounds of Formula I are neuroprotective agents that elicit neuroprotective effects via either a GABA A -dependent mechanism or a GABA A - independent mechanism.
  • Compounds of Formula I demonstrated significant neuroprotection in excitotoxic models of neurodegeneration and in the OGD model provided neuroprotection when administered 6 hours after OGD insult.
  • compounds of Formula I are described as central nervous system (CNS) bioavailable due to their ability to cross the blood-brain barrier (BBB).
  • CNS central nervous system
  • BBB blood-brain barrier
  • GN-28 and GN-28 are described as central nervous system (CNS) bioavailable due to their ability to cross the blood-brain barrier
  • BBB BBB
  • MZ derivatives crossed the blood brain barrier, with a superior brain/plasma ratio for GN-28, but with a higher concentration of free drug observed in the brain of animals treated with GN-38 (Table 1).
  • Compounds of Formula I are characterized as having reduced sedative effects in vivo. Therefore, provided herein is another embodiment of administering the compounds of Formula I to a subject, wherein the compounds of Formula I act in a neurorestorative and/or neuroprotective manner with reduced sedative effects. In another embodiment, provided herein is a method of administering GN- 28 or GN-38 to a subject, wherein GN-28 or GN-38 act in a neurorestorative and/or neuroprotective manner with reduced sedative effects.
  • chloromethiazole (CMZ) caused significant sedation at 10 min and 30 min after drug administration
  • GN-28 caused significant sedation only at 10 min and data for GN-38 did not reach significance (Figure 9).
  • GT1061 4-methyl-5-(2-nitroxyethyl)thiazole (GT1061) is described as a nomethiazole and a NO-mimetic
  • GT1061 is in the hydrochloride salt form. In another embodiment, GT1061 is in the maleate salt form.
  • the hydrochloride salt of GT1061 is shown to elicit signaling through
  • one aspect provided herein is a method of upregulating or activating NO/cGMP/CREB pathway, wherein the method comprises administering GT1061, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
  • the hydrochloride salt of GT1061 only shows sedative effects at higher doses as compared to chloromethiazole (CMZ). Therefore, provided herein is a method of administering GT1061, or a pharmaceutically acceptable salt thereof, wherein GT1061 elicits neuroprotective and/or neurorestorative effects without causing sedation.
  • the dose used to produce a transient sedative effect for the hydrochloride salt of GT1061 was over fifty times the acute dose sufficient to produce procognitive effects.
  • the hydrochloride salt of GT1061 reverses memory deficits in wild type (WT) animals induced by diverse amnestic agents. Therefore, provided herein is a method of administering GT1061, or a pharmaceutically acceptable salt thereof, wherein GT1061 elicits upregulation of the NO/cGMP/CREB pathway to provide neuroprotective and/or neurorestorative effects. Scopolamine resulted in a decrease in latency (98.1+16.6 s) that the hydrochloride salt of GT1061 reversed after either i.p. administration of 1 mg/kg 20 m prior to training (240.7+25.3 s) or
  • the hydrochloride salt of GT1061 is described as having action during the memory consolidation phase.
  • the hydrochloride salt of GT1061 reversed the memory deficit when given up to 90 m after treatment (+30 m: 243.0+34.0 s; +60 m: 300.0+0.0 s; +90: 228.1+71.9 s).
  • the hydrochloride salt of GT1061 increases synaptic plasticity in APP/PS1 mice.
  • the hydrochloride salt of GT1061 resulted in a substantial increase in fEPSP after LTP induction compared to untreated transgenics, to levels identical, and to WT control.
  • the hydrochloride salt of GT1061 shows a trend toward improved memory in APP/PS 1 mice.
  • RAWM a trend towards increased performance over untreated transgenics was observed that did not reach significance
  • the hydrochloride salt of GT1061 decreases inflammation in APP/PS1 mice.
  • Levels of TNF-a, a pro -inflammatory cytokine, were measured using ELISA from total brain homogenates and compared to total protein determined by BCA Figure 17A).
  • APP/PS 1 transgenic animals showed significantly higher levels than WT littermate controls (25.6+2.1, 35.4+2.6 pg/mg protein), and 10-week treatment with the hydrochloride salt of GT1061 reduced this level by 27% (25.9+2.2 pg/mg protein).
  • the hydrochloride salt of GT1061 lowers soluble ⁇ in in APP/PS 1 mice. Therefore, provided herein is a method of administering GT1061 , or a pharmaceutically acceptable salt thereof, wherein GT1061 elicits upregulation of the NO/cGMP/CREB pathway to lower the levels of ⁇ protein in the brain of a subject. ⁇ plaques were assessed using Thioflavin S staining of coronal sections with fluorescent detection ( Figure 17).
  • the hydrochloride salt of GT1061 increases synaptic plasticity in 3xTg mice.
  • the hydrochloride salt of GT1061 was shown to results in more significantly more response to theta bursts, to levels slightly higher than even WT animals, with the hydrochloride salt of GT1061+0DQ treated animals showing a minimal response identical to untreated transgenics.
  • the hydrochloride salt of GT1061 reverses cognitive deficits in 3xTg mice.
  • untreated transgenic showed a latency of 97.1+41.1 s, significantly worse than GT1061 (as the HC1 salt)-treated animals that showed a latency of 247.6+35.2 s (Figure 19).
  • the hydrochloride salt of GT1061 raises pCREB and BDNF, and trends toward lower TNF-oc in 3xTg mice. Biochemical analysis was performed on the brain homogenates after PBS perfusion, and TNF-a showed a trend towards decrease in the hydrochloride salt of GT1061 groups that did not reach significance (2.88+0.20, 2.184+0.34 pg/mg protein, Figure 20A).
  • the hydrochloride salt of GT1061 reduces tau levels in 3xTg mice with negligible effects on ⁇ plaques. Therefore, provided herein is a method of administering GT1061, or a pharmaceutically acceptable salt thereof, wherein
  • GT1061 elicits upregulation of the NO/cGMP/CREB pathway to lower the levels of tau in the brain of a subject.
  • Levels of tau assessed immunohis to chemically using AT8 (1 :1000), an antibody against phosphor ylated tau, showed a significant decrease in the CA1 region of the hydrochloride salt of GT1061 treated animals ( Figure 21 A).
  • a method for treating a neurodegenerative disease comprising administering GT1061, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein GT1061 does not cause sedative effects.
  • AD Alzheimer's disease
  • AD related disorder comprising administering GT1061, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, wherein GT1061 does not cause sedative effects.
  • the treatment comprises the induction of AD, an AD related disorder, or other neurodegenerative disorder, followed by the activation of the compound of the invention, which would in turn diminish or alleviate at least one symptom associated or caused by the disease or disorder being treated.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • preventing means that if the compounds of the present invention are administered to a subject who does not now have the disease but who would normally get the disease or be at increased risk for the disease, they will not get the disease.
  • preventing also includes delaying the development of the disease in an individual who will ultimately get the disease or would be at risk for the disease. By delaying the onset of the disease, the compounds of the invention have prevented the individual from getting the disease during the period in which the individual would normally have gotten the disease or reduce the rate of development of the disease or some of its effects but for the administration of the compounds of the invention up to the time the individual ultimately gets the disease.
  • Preventing also includes administration of the compounds of the invention to those individuals thought to be predisposed to the disease due to familial history and/or due to the presence of one or more biological markers for the disease such as a known genetic mutation of APP or by analysis of APP cleavage products in body tissues or fluids.
  • subject is intended to include organisms (e.g., prokaryotes and eukaryotes) which are capable of suffering from or afflicted with a disease, disorder or condition associated with the activity of a protein kinase.
  • subjects include mammals (e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals).
  • the subject is a human, (e.g. , a human suffering from, at risk of suffering from, or potentially capable of suffering from) AD, an AD related disorder, or other neurodegenerative disorder.
  • the subject is a cell.
  • a “pharmaceutically acceptable salt” includes an acid addition salt and a base addition salt.
  • the acid addition salt includes an inorganic acid salt such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, or an organic acid salt such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, para- toluenesulfonate
  • the base addition salt includes an inorganic base salt such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or an organic base salt such as triethyl ammonium salt, triethanol ammonium salt, pyridinium salt, diisopropyl ammonium salt, and further, an amino acid salt including a basic or acidic amino acid such as arginine, aspartic acid, or glutamic acid.
  • alkyl refers to a fully saturated branched or unbranched hydrocarbon moiety.
  • the alkyl comprises 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbons, 1 to 4 carbons, or 1 to 3 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, so-propyl, «-butyl, sec-butyl, iso-butyl, tert-b tyl, «-pentyl, isopentyl, neopentyl, «-hexyl, 3- methylhexyl, 2,2-dimethylpentyl, 2,3 -dime thylpentyl, «-heptyl, «-octyl, «-nonyl, n- decyl and the like.
  • C x -C y -alkyl indicates a particular alkyl group (straight- or branched-chain) of a particular range of carbons.
  • Ci-C4-alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.
  • alkenyl alone or in combination refers to a straight-chain, cyclic, or branched hydrocarbon residue comprising at least one olefinic bond and the indicated number of carbon atoms.
  • Preferred alkenyl groups have up to 8, preferably up to 6, particularly preferred up to 4 carbon atoms.
  • Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl, 1-cyclohexenyl, 1 -cyclopentenyl.
  • alkynyl includes unsaturated aliphatic groups analogous in length to the alkyls described above, but which contain at least one triple bond.
  • alkynyl includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups.
  • alkynyl further includes alkynyl groups that include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C 2 -C6 for straight chain, C3-C6 for branched chain).
  • C 2 -C6 includes alkynyl groups containing 2 to 6 carbon atoms.
  • cycloalkyl refers to saturated or unsaturated monocyclic, bicyclic, or tricyclic hydrocarbon groups of 3-12 carbon atoms, preferably 3-9, or 3-7 carbon atoms.
  • exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl and the like.
  • Exemplary bicyclic hydrocarbon groups include bornyl, indyl, hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo [2.2.1 Jheptyl, bicyclo[2.2.1]heptenyl, 6,6- dimethylbicyclo [3.1.1 Jheptyl, 2,6,6-trimethylbicyclo [3.1.1 Jheptyl, bicyclo[2.2.2]octyl and the like.
  • Exemplary tricyclic hydrocarbon groups include adamantyl and the like.
  • cycloalkenyl refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 3 rings and 4 to 8 carbons per ring. Exemplary groups include cyclobutenyl, cyclopentenyl, and cyclohexenyl.
  • cycloalkenyl also includes bicyclic and tricyclic groups in which at least one of the rings is a partially unsaturated, carbon-containing ring and the second or third ring may be carbocyclic or heterocyclic, provided that the point of attachment is to the cycloalkenyl group.
  • Alkoxy refers to those alkyl groups, having from 1 to 10 carbon atoms, attached to the remainder of the molecule via an oxygen atom. Alkoxy groups with 1 - 8 carbon atoms are preferred.
  • the alkyl portion of an alkoxy may be linear, cyclic, or branched, or a combination thereof. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, butoxy, cyclopentyloxy, and the like.
  • An alkoxy group can also be represented by the following formula: -OR 1 , where R 1 is the "alkyl portion" of an alkoxy group.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and from one to five heteroatoms, more preferably from one to three heteroatoms, selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroalkyl group is attached to the remainder of the molecule through a carbon atom or a heteroatom.
  • alkylcarbonyl refers to a group having the formula -C(0)-R u , wherein R" is an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties.
  • An "alkylcarbonyl” group can be attached to the remainder of the molecule via an alkyl group (i.e. , -alkyl-C(O)- R a ).
  • alkoxycarbonyl refers to a group having the formula -C(0)0-R m , wherein R m is an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties.
  • alkoxycarbonyl can be attached to the remainder of the molecule via an alkyl group (i.e., -alkyl-C(0)0-R m ).
  • heteroalkylcarbonyl refers to a group having the formula -C(0)R 1V , wherein R 1V is a heteroalkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties.
  • heteroalkylcarbonyl can be attached to the remainder of the molecule via an alkyl or heteroalkyl group (i.e., -alkyl-C(0)0-R iv or -heteroalkyl-C(0)0-R iv ).
  • aryl includes aromatic monocyclic or multicyclic e.g., tricyclic, bicyclic, hydrocarbon ring systems consisting only of hydrogen and carbon and containing from six to nineteen carbon atoms, or six to ten carbon atoms, where the ring systems may be partially saturated.
  • Aryl groups include, but are not limited to, groups such as phenyl, tolyl, xylyl, anthryl, naphthyl and phenanthryl.
  • Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
  • heteroaryl represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
  • heteroaryl is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.
  • heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no hetero atoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively.
  • heterocycle refers to a five-member to ten- member, fully saturated or partially unsaturated nonaromatic heterocylic groups containing at least one heteroatom such as O, S or N.
  • heteroatom such as O, S or N.
  • the most frequent examples are piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl or pirazinyl. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom.
  • alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkoxy, aryl, heteroaryl, and heterocycle groups described above can be "unsubstituted” or “substituted.”
  • substituted is intended to describe moieties having substituents replacing a hydrogen on one or more atoms, e.g. C, O or N, of a molecule.
  • substituents can independently include, for example, one or more of the following: straight or branched alkyl (preferably C 1 -C 5 ), cycloalkyl (preferably C3-C8), alkoxy (preferably Ci-Ce), thioalkyl (preferably Ci-Ce), alkenyl (preferably C 2 -C6), alkynyl (preferably C 2 -C6), heterocyclic, carbocyclic, aryl ⁇ e.g. , phenyl), aryloxy ⁇ e.g. , phenoxy), aralkyl ⁇ e.g. , benzyl), aryloxyalkyl ⁇ e.g.
  • phenyloxyalkyl arylacetamidoyl, alkylaryl, heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group, heteroarylcarbonyl, or heteroaryl group, (CR'R")o- 3 NR'R"
  • amine or “amino” should be understood as being broadly applied to both a molecule, or a moiety or functional group, as generally understood in the art, and may be primary, secondary, or tertiary.
  • amine or “amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon, hydrogen or heteroatom.
  • the terms include, for example, but are not limited to, "alkyl amino,” “arylamino,” “diarylamino,” “alkylarylamino,” “alkylaminoaryl,” “arylaminoalkyl,” “alkaminoalkyl,” “amide,” “amido,” and “aminocarbonyl.
  • alkyl amino comprises groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • arylamino and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively.
  • alkylarylamino
  • alkylaminoaryl or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group.
  • alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
  • amide includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group.
  • alkaminocarbonyl or
  • alkylaminocarbonyl groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group.
  • alkylaminocarbonyl alkenylaminocarbonyl
  • alkynylaminocarbonyl alkynylaminocarbonyl
  • arylaminocarbonyl alkylcarbonylamino
  • alkenylcarbonylamino alkenylcarbonylamino
  • alkynylcarbonylamino and “arylcarbonylamino” are included in term “amide.” Amides also include urea groups (aminocarbonylamino) and carbamates
  • the term "amine” or “amino” refers to substituents of the formulas N(R 8 )R 9 , CH 2 N(R 8 )R 9 and CH(CH 3 )N(R 8 )R 9 , wherein R 8 and R 9 are each, independently, selected from the group consisting of H and (Ci-C4-alkyl)o-iG, wherein G is selected from the group consisting of COOH, H, P0 3 H, SO 3 H, Br, CI, F, 0-Ci_4-alkyl, S-Ci_ 4 -alkyl, aryl, C(0)OCi-C 6 -alkyl, C(0)C C 4 -alkyl-COOH, C(0)Ci-C 4 -alkyl and C(0)-aryl.
  • the compounds of this invention may include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates) are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Compounds described herein may be obtained through art recognized synthesis strategies.
  • substituents of some of the compounds of this invention include isomeric cyclic structures. It is to be understood accordingly that constitutional isomers of particular substituents are included within the scope of this invention, unless indicated otherwise.
  • tetrazole includes tetrazole, 2H-tetrazole, 3H-tetrazole, 4H-tetrazole and 5H-tetrazole.
  • Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids (e.g., hydrochloric, hydrobromic, sulphuric, or phosphoric acids) and organic acids (e.g., succinic, malaeic, acetic, or fumaric acid).
  • inorganic acids e.g., hydrochloric, hydrobromic, sulphuric, or phosphoric acids
  • organic acids e.g., succinic, malaeic, acetic, or fumaric acid.
  • non-pharmaceutically acceptable salts e.g., oxalates
  • solvates and hydrates of the invention are also included within the scope of the invention.
  • the conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base (e.g., sodium carbonate or potassium hydroxide), to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.
  • base e.g., sodium carbonate or potassium hydroxide
  • Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fuma
  • acids such as oxalic acid, which cannot be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt.
  • Metal salts of a chemical compound of the invention include alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group.
  • In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform.
  • Suitable bases include triethylamine or pyridine.
  • compounds of the invention having a free carboxy group can be esterified using standard conditions which can include activation followed by treatment with the desired alcohol in the presence of a suitable base.
  • diastereoisomers can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallisation and/or chromatographic separation, for example over silica gel or by medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallisation, or by chromatography over optically active column materials.
  • Intermediates and final products can be worked up and/or purified according to standard methods, e.g. , using chromatographic methods, distribution methods, (re-) crystallization, and the like.
  • mixtures of isomers that are formed can be separated into the individual isomers, for example diastereoisomers or enantiomers, or into any desired mixtures of isomers, for example racemates or mixtures of diastereoisomers, for example analogously to the methods described in Science of Synthesis: Houben-Weyl Methods of Molecular Transformation. Georg Thieme Verlag, Stuttgart, Germany, 2005.
  • solvents from which those solvents that are suitable for any particular reaction may be selected include those mentioned specifically or, for example, water, esters, such as lower alkyl-lower alkanoates, for example ethyl acetate, ethers, such as aliphatic ethers, for example diethyl ether, or cyclic ethers, for example
  • liquid aromatic hydrocarbons such as benzene or toluene
  • alcohols such as methanol, ethanol or 1- or 2-propanol
  • nitriles such as acetonitrile
  • halogenated hydrocarbons such as methylene chloride or chloroform
  • acid amides such as dimethylformamide or dimethyl acetamide
  • bases such as heterocyclic nitrogen bases, for example pyridine or N-methylpyrrolidin-2-one
  • carboxylic acid anhydrides such as lower alkanoic acid anhydrides, for example acetic anhydride
  • cyclic, linear, or branched hydrocarbons such as cyclohexane, hexane, or isopentane, or mixtures of those solvents, for example aqueous solutions, unless otherwise indicated in the description of the processes.
  • solvent mixtures may also be used in working up, for example by chromatography or partitioning.
  • the compounds, including their salts, may also be obtained in the form of hydrates, or their crystals can, for example, include the solvent used for
  • the invention relates also to those forms of the process in which a compound obtainable as an intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in a protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in situ.
  • the compounds described herein can be prepared using synthetic procedures described in, for example, International Patent Application No. WO 2011/140,198, filed November 10, 2011.
  • the compound GT1061, and its pharmaceutically acceptable salt forms can be prepared using synthetic procedures described in, for example, U.S. Patent Nos. 5,807,847; 5,883,122; and 6,310,052. Each of these patent documents are incorporated herein by reference in their entireties.
  • Scheme 1 demonstrates the preparation of compounds of Formula I, such as compounds GN-27, 28, 36, 38, 40, and 43 as referenced herein.
  • MZ derivatives were initially screened at a single 50 uM dose in rat primary cortical neurons (10-12 DIV) subjected to OGD with analysis of cell viability by MTT assay.
  • the 50 uM dose was chosen to maximize protection, and no MZ toxicity was observed for neuroprotective analogues even at double this dose (Figure IB).
  • a subset of structural compounds with neuroprotective activity equal to or greater than CMZ were subjected to insult with or without picrotoxin, a GABA A channel blocker, at 100 uM, a dose sufficient to eliminate signaling through the
  • GABA A receptor without toxicity, with either blockers or vehicle applied one hour before the start of insult and continued throughout. Muscimol, a potent, selective direct agonist of GABA A with demonstrated neuroprotection against excitotoxic insult, was included to provide an example of a pure MOA.
  • MZ neuroprotective agents were selected in both of which the methylthiazole ring of CMZ was preserved: all were neuroprotective against OGD with equivalent or superior efficacy to CMZ, and assay with use of picrotoxin to block the GABA A chloride channel revealed varied dependence on GABA A signaling ( Figure 3).
  • MZ derivatives GN-0, GN-46, and GN- 38 were neuroprotective, but with no significant loss of efficacy on picrotoxin co- treatment.
  • MZ derivatives, GN-12, GN-28, GN-35, GN-36, and GN-37 showed significant attenuation by picrotoxin.
  • Muscimol a selective, potent GABA A receptor agonist, was used as a positive control at equimolar concentration to both CMZ and MZ derivatives.
  • MZ derivatives were tested against neurotoxicity resulting from application of oligomeric full-length human Ar31-42.
  • MZ derivatives 50 ⁇
  • oligomeric Af3i_42 250 nM
  • All MZ derivatives tested were observed to deliver significant neuroprotection relative to the vehicle control ( Figure 4).
  • picro toxin was used to estimate the contribution of GABA A receptors to cell survival.
  • OGD OGD
  • GN-28 and GN-38 were selected for more in-depth study as representatives of neuroprotective MZ derivatives with greater and lesser dependence on GABA A pathways, respectively ( Figure 2).
  • Dose ranging in the OGD assay showed that CMZ and novel compounds had negligible effect on cell survival in the absence of OGD at doses of 100 nM - 100 ⁇ , results consistent with previous observations on CMZ, and furthermore, no artifactual interaction was found between any novel compounds and MTT (Figure 1).
  • Concentration-response curves for GN-28 and GN-38 100 nM - 100 ⁇
  • Neuroprotection by both MZ derivatives was observed at concentrations equivalent to those known to be pharmacologically relevant for CMZ.
  • OGD is a model of ischemia-reperfusion injury. Neuroprotective mechanisms elicited during cellular hypoxia and ischemia can differ from those that provide protection upon reoxygenation and later apoptotic and inflammatory mechanisms. Preconditioning provides a further array of mechanisms for protection against subsequent insult.
  • GN- 28 or GN-38 were added at four different timepoints: i) 1 h pretreatment with removal of drug at commencement of OGD (Pre-tx); ii) immediately at the start of OGD (+0 h); iii) immediately after the 2 h OGD period (+2 h); and iv) 6 hours after start of OGD (+6 h).
  • OGD was transient for 2h in each paradigm. Both GN-28 and GN-38 showed a similar protection profile, with no significant effect seen on pretreatment, but efficacy was maintained up to six hours after initiation of OGD and four hours after initiation of oxygen/glucose reperfusion (Figure 5B).
  • Example 11 Protection Against NMD A or Glutamate Toxicity Variably Involves GABA A
  • GN-38 in contrast, was neuroprotective, but the effects of picrotoxin were not significant against either NMDA or glutamate insult.
  • Concentration- response curves for cell survival were normalized to vehicle treated cells within the same plate either subject to excitotoxic insult (0% survival) or not (100% survival).
  • concentration-response curves were similar ( Figure 6B, 7B). Observations suggest that GN-38 does not depend on the GABA A receptor for neuroprotective activity against excitotoxic insult, however, significant neuroprotection was elicited against pure glutamate receptor mediated excitotoxicity.
  • Example 12 Neuroprotection Against High Dose Afl via GABA A -independent Mechanism
  • Table 1 Plasma & brain concentrations of MZ compounds determined 20 m after i.p. administration.
  • Example 16 GT1061 shows sedative effect at higher doses than CMZ.
  • CMZ showed a significant deficit (35.0+12.8 s), while the hydrochloride salt of GT1061 was similar to vehicle (93.5+5.4, 115.8+6.3 s), and by 60 m, the hydrochloride salt of GT1061 and vehicle were nearly identical (122.1+10.7 s, 109.1+7.0 s), while CMZ had a latency to fall of only 73.0+6.5 s.
  • the dose used to produce a transient sedative effect for GT1061 was over fifty times the acute dose sufficient to produce procognitive effects.
  • Loss of righting reflex is defined as failure to place four paws on the ground within 30 s after placing the mouse on its back, and the duration of this loss of reflex from time of administration was measured every 2 m for 2 h post administration. No substantial LORR was observed for either compound until 125 mg/kg, when CMZ treated animals showed a LORR for 23.8+2.7 m, at which dose the hydrochloride salt of GT1061 only showed a LORR for 4.8+1.7 m.
  • GT1061 continued to show less sedation than CMZ until 175 mg/kg (GT: 90.5+20.9 m, CMZ: 66.2+7.7), an amount almost 200 times the acute dose necessary for procognitive effect.
  • Example 17 GT1061 reverses memory deficits in WT animals induced by diverse amnestic agents.
  • the hydrochloride salt of GT1061 was tested at 1 mg/kg using male C57B1/6 mice in the step through passive avoidance (STPA) behavioral model of memory, where mice are trained to associated a mild electric shock (0.5 mA) with the dark side of a light-dark box and latency to enter is assessed 24 h after training ( Figure 12).
  • STPA passive avoidance
  • a variety of compounds were used at doses known to induce memory deficits given 20 m before training, including scopolamine (1 mg/kg), a muscarinic receptor antagonist; MK-801 (0.1 mg/kg), an NMDA receptor antagonist; diazepam (0.5 mg/kg), a direct GABA A receptor agonist; and L-NAME (50 mg/kg), a non-specific NOS receptor antagonist.
  • L-NAME caused a latency of 22.0+10.4 s, which was not completely reversible by GT1061 at 20 minutes prior to training (77.7+17.5 s), but was reversed when GT1061 was given 50 minutes prior to training (21 1.7+31.4 s).
  • Example 18 GT1061 has action during memory consolidation phase.
  • GT1061 When administered as a bolus prior to training, GT1061 was effective when given within 40 m of the start of training, while not effective when given 1 or 2 h prior (-20 m: 134.0+11.3 s; -60 m: 125.2+37.1 s; -40 m: 263.0+17.1 s; -20 m: 280.0+11.4 s). When given after training is complete, GT1061 reversed the memory deficit when given up to 90 m after treatment (+30 m: 243.0+34.0 s; +60 m: 300.0+0.0 s; +90: 228.1+71.9 s).
  • Example 19 GT1061 increases synaptic plasticity in APP/PS1 mice.
  • the hydrochloride salt of GT1061 resulted in a substantial increase in fEPSP after LTP induction compared to untreated transgenics, to levels identical to WT control.
  • Example 20 GT1061 shows a trend towards improved memory in APP/PS1 mice.
  • the hydrochloride salt of GT1061 was also given to APP/PS1 mice and WT controls at 20 mg/kg/day in hydrogel (drinking water substitute) + 1 mg/kg i.p. injection (to ensure a minimal amount of delivery) for 10 weeks starting at 2 months of age.
  • the animals were tested in the radial arm water maze (RAWM) and then immediately sacrificed, with blood and brain tissue collected after PBS perfusion for biochemical and immunohis to chemical analysis.
  • RAWM radial arm water maze
  • Example 21 GT1061 decreases inflammation in APP/PS1 mice.
  • TNF-a a pro-inflammatory cytokine
  • APP/PS1 transgenic animals showed significantly higher levels than WT littermate controls (25.6+2.1 , 35.4+2.6 pg/mg protein), and 10- week treatment with GT1061 reduced this level by 27% (25.9+2.2 pg/mg protein).
  • Iba-1 (1 :1000) staining of coronal slices showing marked reductions in activated microglia in both the hippocampus and cortex of GT1061(as the HC1 salt)-treated mice ( Figure 16B).
  • LaFerla's 3xTg transgenic mouse model (APPsweKooNimoPS 1 ml461, iL,taup3olL) was chosen for its inclusion of tau pathology.
  • the hydrochloride salt of GT1061 (50 uM) was added to aCSF perfusate at least 30 m prior to induction of LTP and continued throughout. LTP was induced using ten theta bursts, and the resulting fEPSP were recorded in the CA1 area for -50 m ( Figure 19A).
  • the hydrochloride salt of GT1061 again resulted in a substantial increase in fEPSP after LTP induction compared to untreated transgenics, to levels similar to WT control.
  • ODQ soluble guanylyl cyclase
  • Example 24 GT1061 reverses cognitive deficits in 3xTg mice.
  • 3xTg mice were given the hydrochloride salt of GT1061 at doses identical to the APP/PS1 study (20mg/kg/day in hydro gel + 1 mg/kg i.p. injection) for 10 weeks starting at 12 months of age for females and 15 months of age for males (to account for delayed pathology in males). After completion of the trial, the animals were tested using
  • Example 25 GT1061 raises pCREB and BDNF, and trends towards lowered TNF- a in 3xTg mice.
  • the end product of NO/cGMP/sGC activation is thought to be pCREB, so pCREB levels in whole brain homogenates were assayed using ELISA and found to be increased more than 2-fold in GT1061 (as the HC1 salt form)-treated animals (0.424+0.031 , 1.066+0.106 units/mg protein; Figure 20B), while BDNF, a downstream target of pCREB, showed a trend towards increase that did not reach significance (129.4+8.1, 158.1+11.2 pg/mg protein).
  • Example 26 GT1061 reduces tau levels in 3xTg mice with negligible effect on Afiplaques.
  • Double transgenic mice (APP/PS1) expressing both the human APP (K670M:N671L) and PS 1 (M146L-line 6.2) mutations were compared with wild type littermates.
  • LaFerla triple transgenic mice (3xTg AD) expressing human APP (K670N; M671L), PS1(M146V) and tau (P301L) were generated from breeding pairs provided by Dr. F. LaFerla in Dr. Mufson group of Rush
  • cortices were dissected from the brains of Embryonic Day 16 (E16-18) Sprague-Dawley rats (Charles River Laboratories) fetuses. The embryos were transferred to a plate with L15 medium (Leibovitz, Sigma L5520) and the brains were isolated and the cortices were dissected.
  • L15 medium Leibovitz, Sigma L5520
  • the cortices were transferred to another plate with 1 ml of plating medium[(Basal medium eagle lx with 10% Horse serum, 10% Fetal Bovine Serum, and 10% GGG (Dextrose, L-Glutamine 200mM, and Gentamicine)] where they were dissociated by repeated passage through a series of fire polished constricted Pasteur pipettes. Then the supernatant was transferred to a new tube and the cells were plated at a concentration of 10 6 cells/ml on poly-L-lysine coated plates and maintained in a humidified incubator (37°C with air and 5% C02).
  • the plating medium was replaced by the growing medium (Neurobasal medium 100ml , B27 supplement minus AO 2ml, L-Glutamine (200mM) 250uL), then 3 days later half the growing media was replaced by fresh growing media, and the cells were left till they were 8-9 days old were they were used for experiments.
  • Soluble oligomers of ⁇ 42 were prepared as previously described. Briefly, lyophilized full-length human- sequence peptide was dissolved in 1 ,1 ,1,3,3,3- Hexafluoro-2-propanol and evaporated to leave a peptide film, which was then dissolved to 5 mM in DMSO and added to cold phenol-free F-12 cell culture media and allowed to stand 24 h at 4°C. This procedure reliably develops soluble oligomers upon addition to culture media as confirmed by atomic force microscopy [388]. After 10-1 1 DIV, cultures were resupplied with fresh growth media and oligomeric ⁇ was added at a final concentration of 250 nM or 5 uM.
  • Picrotoxin blockade was performed as in OGD and added 1 h before oligomers. One hour after addition of oligomers, compounds were added at 50 uM. Cultures were incubated for 4 days, after which time cell survival was assayed by MTT as above.
  • mice were rapidly decapitated, and brains were removed into an ice-cold aCSF solution (in mM: NaCl 124, KC1 3.0, KH2P04 1.25, NaHC03 25.7, D-glucose 10, L-ascorbate 2.0, MgS04 2.5, and CaC12 3.3) and sectioned on a tissue chopper into 400 um sections. Slices were transported to a 37°C solution of aCSF, continuously bubbled with 95% 0 2 / 5 % C0 2 , and allowed to recover at least 60 m before experiment.
  • aCSF solution in mM: NaCl 124, KC1 3.0, KH2P04 1.25, NaHC03 25.7, D-glucose 10, L-ascorbate 2.0, MgS04 2.5, and CaC12 3.3
  • stimulus intensity was set to evoke a submaximal fEPSP and continuously monitored at 20 s intervals for at least 15 m to establish a stable baseline.
  • SERMs 100 nM
  • G15 100 nM
  • LTP was induced using a theta burst induction protocol by applying 10 bursts of four pulses at 100 Hz with an interburst interval of 200 msec.
  • Resulting fEPSP was monitored at 20 s intervals for 60 m post TBS.
  • mice Male C57B1/6 mice (Charles River's Laboratory) or eNOS KO animals. Animals were injected (ip) with 2 mg/kg drug 1 h before sacrifice. After euthanasia via CO 2 , PBS was perfused through the left ventricle, and cortices, hippocampi and plasma were collected.
  • Tissue samples were weighed and pulverizing using a mortar and pestle in liquid nitrogen. To extract NO from tissue or plasma, samples were washed 2x with 0.5 mL deionized water followed by filtration through a 10,000 MWCO. The supernatant (deproteinized) was analyzed by chemiluminescence with SIEVERS
  • SERMs were injected (ip) 1 h before blood collection, and L-NAME was injected (ip) 30 m before blood collection.
  • the mouse was sedated by exposing it to CO 2 until unconscious and then the blood was collected in 4.5 mL BD
  • Vacutainer Glass Evacuated Blood Collection Tubes (with 0.105 M Buffered Sodium Citrate) by cardiac puncture.
  • the blood samples were centrifuged at 300g for 30 m to separate the plasma from the blood, and then 200 uL of plasma was taken from each sample and the PT and APTT were measured using an ACL 7000 Coagulation analyzer.
  • the analyzer injects 125 uM of PT-fibrinogen (lyophilized rabbit brain calcium thromboplastin with stabilizers, polybrene, buffer and preservatives) to 75 uL of plasma sample to measure the PT, and 75 uL of synthAFAX (0.025 M) + 100 uL of CaCl 2 (0.02 M) were added to 75 uL of plasma to measure APTT.
  • PT-fibrinogen lyophilized rabbit brain calcium thromboplastin with stabilizers, polybrene, buffer and preservatives
  • APP/PS1 mice Six- week-old APP/PS1 mice and same age wild type littermates were grouped into different drug or vehicle treatment. Each animal was given assigned drug (equimolar to lmg/kg of GT-1061) via i.p. injections once a day until the end of the study (total 9-12 weeks of treatment until the behavioral tests finished at age of week 16-18). APP/PS1 mice were also administered with the same compound
  • mice (equimolar to 20mg/kg/day of GT-1061) in 0.5 % Tween80 drinking water. 3xTg mice were given 20mg/kg/day of GT-1061 in hydrogel to replace drinking water.
  • RAWM Radial-arm water maze
  • the RAWM task has proven informative in the analysis of short-term memory of other transgenic AD models.
  • the RAWM consisted of a tank filled with opaque water by non-toxic white paint. Walls were positioned so as to produce 6 arms, radiating from a central area. Spatial cues were present on the walls of the testing room. At the end of one of the arms there was a clear 10 cm submerged platform that remained in the same location for every trial on a given day, but was moved about randomly from day to day.
  • the mouse could not use its long-term memory of the location of the platform on previous days, but had to rely on the short-term memory of its location on the day in question based on spatial cues that were present in the room.
  • Each trial lasted 1 min and errors were counted each time the mouse entered the wrong arm or needed more than 10 sec to reach the platform. After each error the mouse was pulled back to the start arm for that trial. After 4 consecutive acquisition trials, the mouse was placed in its home cage for 30 min, then returned to the maze and administered a 5th retention trial. Testing was considered completed when the WT mice made the same number of errors during the 4th and 5th trial.
  • Levels of brain ⁇ 2, TNF-a, pCREB (Biosource) and BDNF (Promega) were determined by sandwich ELISA kit according to the manufacturer's Protocol) of hemi-brains homogenized in cell extraction. Buffer supplied with protease inhibitors and PMSF, and the insoluble pellets were then extracted in guanidine buffer. Briefly, the frozen hemi-brain tissues were homogenized in five volumes of carbonate buffer (lOOmM sodium carbonate, 50mM NaCl , containing protease inhibitors, pH 10) or Cell Extraction Buffer (Invitrogen, supplied with protease inhibitors and PMSF) and centrifuged at 20,000xg for 20 minutes at 4°C.
  • carbonate buffer la OmM sodium carbonate, 50mM NaCl , containing protease inhibitors, pH 10
  • Cell Extraction Buffer Invitrogen, supplied with protease inhibitors and PMSF
  • This water-soluble supernatant (A) was analyzed for TNF-a, pCREB and BDNF levels using ELISA kits.
  • the pellets were homogenized again in five volumes of 5 M guanidine HC1 made in 50 mM Tris-HCl buffer, pH 8.0, for 1 -2 hours in room temperature.
  • sections were rinsed in phosphate buffer (PB), washed in Tris- buffered saline (TBS ; pH 7.4), incubated in TBS containing sodium meta- periodate (0.1 M; 20 min), rinsed for 30 min in a solution containing TBS and Triton X- 100 (0.25%; TBST) and then blocked in TBST with 3% goat serum for 1 h. Sections were subsequently incubated with primary antibody in TBST containing 1 % goat serum overnight at room temperature with constant agitation.
  • PB phosphate buffer
  • TBS Tris- buffered saline
  • TBS TBS containing 1 % goat serum
  • sections were incubated with secondary antibody (1 :200) in TBS (goat anti-mouse IgG for 6E10 and ATE, or goat anti-mouse IgM for Alz50) with 1 % goat serum at room temperature for 1 h.
  • Sections were washed with TBS and incubated with avidin— biotin complex (1 :500; "Elite Kit,” Vector Labs).
  • Tissue was then washed in sodium acetate trihydrate (0.2 M) and imidazole (1.0 M) solution (pH 7.4 with acetic acid).
  • Reaction products were visualized using an acetate— imidazole buffer containing 0.05% 3/3 ' -diaminobenzidine tetrahydrochloride (DAB; Sigma, MO) and 0.0015% freshly prepared H 2 O 2 . Sections were washed in acetate— imidazole buffer to terminate the histochemical reaction, mounted on to alum-submersed slides, air dried for 24 h, dehydrated through a series of graded alcohols (70%, 95%, and 100%), cleared in xylene, and cover-slipped with DPX. Sections were analyzed at the light microscopic level with the aid of an Olympus microscope.
  • DAB 3/3 ' -diaminobenzidine tetrahydrochloride
  • a rotarod apparatus Rotamex-5 (Columbus instruments, OH), was used to test sedative properties of the test compounds. Animals were placed on a spindle (rod) and latency to fall was detected with a 0.1 second temporal resolution by a series of photocells located above the rotating rod in the apparatus. Mice were trained for 3-5 days using an accelerated mode (4-40 rpm within 2 min) until the mean latency to fall reached to 100 ⁇ 10 s. Each training session started with a 30 s trial on a non-rotating rod, followed by a 60 s rotating at constant speed of 4 rpm. On the test day, after i.p.
  • mice were acclimated on the rod for 30 s (0 rpm) and 60 s (4 rpm), and then mean latencies to fall were recorded using the accelerated mode. The mean latency to fall was compared to that of pre-treatment for each group.

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  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des procédés de traitement, comprenant l'administration d'une quantité efficace d'un composé organique thérapeutique à un sujet en ayant besoin.
PCT/IB2013/001994 2012-07-16 2013-07-16 Composés pour le traitement de la maladie d'alzheimer WO2014013338A2 (fr)

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WO2001049275A2 (fr) * 1999-12-29 2001-07-12 Queen's University At Kingston Procedes et dispositifs destines a reduire la douleur
US20050137191A1 (en) * 1996-06-04 2005-06-23 Thatcher Gregory R. Nitrate esters and their use for mitigating cellular damage
WO2005105065A2 (fr) * 2004-05-05 2005-11-10 Renopharm Ltd. Donneurs d'oxyde nitrique destines au traitement de maladies intestinales inflammatoires
WO2011140198A2 (fr) * 2010-05-05 2011-11-10 The Board Of Trustees Of The University Of Illinois Composés et méthodes permettant de traiter des troubles cérébraux
WO2012027471A1 (fr) * 2010-08-24 2012-03-01 Sgc Pharma, Inc. Composé de sel

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US20050137191A1 (en) * 1996-06-04 2005-06-23 Thatcher Gregory R. Nitrate esters and their use for mitigating cellular damage
WO2000054756A2 (fr) * 1999-03-15 2000-09-21 Queen's University At Kingston Esters de nitrate et leur utilisation dans le traitement d'etats neurologiques
WO2001049275A2 (fr) * 1999-12-29 2001-07-12 Queen's University At Kingston Procedes et dispositifs destines a reduire la douleur
WO2005105065A2 (fr) * 2004-05-05 2005-11-10 Renopharm Ltd. Donneurs d'oxyde nitrique destines au traitement de maladies intestinales inflammatoires
WO2011140198A2 (fr) * 2010-05-05 2011-11-10 The Board Of Trustees Of The University Of Illinois Composés et méthodes permettant de traiter des troubles cérébraux
WO2012027471A1 (fr) * 2010-08-24 2012-03-01 Sgc Pharma, Inc. Composé de sel

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